Mass spectrometry strategies for comprehensive lipidome analysis of colorectal cancer cells and their secreted exosomes

Lipids are essential for numerous cellular functions and disruption of lipid metabolism or signaling has been demonstrated to be associated with the onset and progression of diseases, including cancer. Therefore, further investigation into the details of lipid alterations and metabolism in cancer may be beneficial in biomarker and cancer metabolic discovery. Exosomes, 40-100nm vesicles secreted from many cells, have been implicated in cellular communication and progression and metastasis of cancer. However, characterization of exosome lipidomes and their potential roles in cancer progression have had little exploration. Several methods for lipid analysis have been developed such as gas chromatography (GC) and liquid chromatography (LC); however, many of these methods are time consuming and offer limited information on individual lipid structural identity. Mass spectrometry has proven to be beneficial as both a sensitive detector following separation by GC or LC and as a standalone strategy for lipid analysis without previous separation. Recent developments in high resolution mass spectrometry enhance the ability for mass spectrometry to be utilized for unambiguous identification of lipid species.;In this dissertation, a 'shotgun' lipidomics strategy consisting of sequential functional group selective chemical modification reactions coupled with high-resolution/accurate mass spectrometry analysis, and 'targeted' tandem mass spectrometry (MS/MS), has been developed and applied toward the comprehensive identification, characterization and quantitative analysis of changes in relative abundances of greater than 1500 individual glycerophospholipid, glycerolipid, sphingolipid and sterol lipids between a primary colon adenocarcinoma cell line, SW480, its metastasized derivative, SW620, and their secreted exosomes. Selective chemical derivatization of phosphatidylethanolamine and phosphatidylserine lipids using a 'fixed charge' sulfonium ion containing reagent, d6-S,S'-dimethylthiobutanoylhydroxysuccinimide ester (d 6-DMBNHS) or 13C1-DMBNHS, eliminates the possibility of isobaric mass overlap of these species with the precursor ions of all other lipids in crude lipid extracts. Subsequent selective mild acid hydrolysis of plasmenyl-ether containing lipids using formic acid or, alternatively, a method involving the use of plasmenyl-ether selective derivatization with iodine and methanol enables these species to be differentiated from isobaric mass plasmanyl-ether containing lipids. Using this approach, statistically significant differences in the abundances of numerous lipid species previously identified as being associated with cancer development, or that play known roles as mediators in a range of physiological and pathological processes were observed among the cells and exosomes. In the cells, increases in several ether-containing glycerophospholipids, triglyceride and cholesterol ester lipid levels in the SW620 metastatic colon cell line lipid extracts were observed compared to the SW480 cells. Increases in ether phospholipids were also observed in the exosomes compared to their respective cell lines as well as overall glycerophosphoethanolamine, glycerophosphoserine and sphingomyelin levels. The information provided by these analysis techniques is expected to lead to a broader knowledge of the role of lipid metabolism and cancer progression.